Nucleoside analogue method

ABSTRACT

A method is provided of making a dideoxynucleoside mono-or tri-phosphate, which is optionally 32-P or 33-P or 35-S radiolabelled which involves reacting the corresponding dideoxynucleoside with an optionally radiolabelled nucleotide phosphate or thiophosphate donor in the presence of a kinase of phosphotransferase enzyme which catalyses the reaction. A method of sequencing nucleic acids by a chain termination of a kinase or phosphotransferase enzyme which catalyses the reaction. A method of sequencing nucleic acids by a chain termination technique involves detecting the products of enzymatic synthesis by means of isotopically labelled chain terminating nucleotide analogues.

This application is filed under 35 USC 371 as the National Stage ofPCT/GB94/02630, filed Dec. 1, 1994, now WO 95/15395, published Jun. 8,1995 and has priority to EPO application Ser. No. 93309597.8 filed Dec.1, 1993.

This application is filed under 35 USC 371 as the National Stage ofPCT/GB94/02630, filed Dec. 1, 1994, now WO 95/15395, published Jun. 8,1995 and has priority to EPO application Ser. No. 93309597.8 filed Dec.1, 1993.

α³² P! dideoxyadenosinetriphosphate is commercially available. Thecurrent chemical production method is very inefficient. This inventionprovides an enzymatic method of preparation, which improves efficiency.The method is applicable to a wide range of compounds besides this one.

T4 polynucleotide kinase (T4 PNK) is usually associated with thephosphorylation of the 5'-OH group of an oligonucleotide, DNA or2-deoxynucleoside-3-monophosphate (or ribo) by transfer of thegamma-phosphate group from ATP. (Analytical Biochemistry 214, 338-340,1993). It is generally believed that for T4 PNK to phosphorylate the5'-OH group of a nucleotide, the nucleotide must contain a 5'-OH and a3'-phosphate group. Because the 3'-phosphate group is clearly absentfrom 2',3'-dideoxynucleosides and other known chain terminators, the useof T4 PNK to catalyse their phosphorylation has hitherto been consideredimpossible. This invention results from our surprising discovery that T4PNK can be used to catalyse this reaction. The invention covers the useof T4 PNK and other enzymes to catalyse this and related reactions.

Known kits for sequencing nucleic acids comprise supplies of all fournucleotides, and supplies of all four 2',3'-dideoxynucleotides, and asupply of one nucleotide which has been labelled, generallyradioactively labelled, to permit detection of the products aftersequencing by electrophoresis. In another aspect, this invention isbased on the realization that improved results can be obtained byradiolabelling the dideoxynucleotides.

Kits containing fluorescently labelled chain terminators (ddNTPs) areknown but isotopically labelled ddNTPs have structures which are lesslikely to interfere with polymerase activity, gel mobility and do notrequire sophisticated equipment for detection.

In one aspect, this invention provides a method of making a nucleotideor nucleotide analogue or nucleotide adduct, having a 5'-phosphate or a5'-thiophosphate group which method comprises reacting a startingnucleoside or nucleoside analogue or nucleoside adduct having a 5'-OHgroup but no 3'-phosphate group with a nucleotide phosphate orthiophosphate donor in the presence of an enzyme which catalyses thereaction. The nucleoside can be an unmodified ribo ordeoxyribonucleoside e.g. 2' deoxyadenosine.

It is possible that the nucleoside, nucleoside analogue or nucleosideadduct may be non-labelled and that the phosphate donor is alsonon-labelled. This produces the corresponding nucleotide without theneed to use chemical phosphorylating agents which may be damaging to thestarting material in some circumstances.

Alternatively the nucleoside can be labelled with a detectable isotopee.g. a radioisotope such as for example ³ H or ¹⁴ C and then convertedto the corresponding labelled nucleotide with a non-labelled phosphatedonor or thiophosphate donor.

Preferably the nucleotide phosphate or thiophosphate donor isradiolabelled with a detectable isotope e.g. a radioactive isotope suchas ³² P or ³³ P or ³⁵ S, whereby the obtained nucleotide or nucleotideanalogue or nucleotide adduct is radiolabelled by virtue of having a5'-phosphate or 5'-thiophosphate group comprising ³² P or ³³ P or ³⁵ S

The term nucleoside analogue refers to a compound which is similar to anucleoside and is capable of performing at least some of the biochemicalfunctions of a nucleoside, and includes monomers and multimers. Therefollows a non-exhaustive list of nucleoside analogues.

Base Modifications

2-aminoadenosine

5-bromocytosine

5-methylcytosine

5-(1-propynyl)cytosine

5-(1-propynyl)uracil

5-aminoallyluracil

5-aminoallyluracil-"label"

thiouracil/thiothymine/thioguanine

aziridene derivatives

Sugar Modifications

2'-O-alkyl (e.g. allyl or methyl)

2'-fluoro

2'-amino

2'-deoxy

3'-deoxy

3'-"label"

3'-fluoro

3'-amino

3'-azido

2',3'-unsaturated

Combinations of base and sugar modifications Phosphate Modifications

Phosphorothioate

Phosphorodithioate

Hydrogen phosphonate

Methyl phosphonate

Phosphotriester

Phosphoramidite

Methylene bridge derivatives

Modified backbones

Polyamide nucleic acid (PNA) modified to give the equivalent of a 5'hydroxyl, e.g. having the formula ##STR1##

(For polyamide nucleic acids (PNA) see P. E. Neilsen, Science Volume254, Dec. 6, 1991, Reports pages 1497-1500). Anti-sense and anti-geneoligonucleotides provide another example of nucleoside analogues.

The preferred and most important nucleoside analogues with which theinvention is concerned are the four 2',3'-dideoxynucleosides ddA, ddC,ddG and ddT.

The term nucleoside adduct refers to a compound which results from theinteraction between reactive entities and DNA or RNA. Such reactiveentities include carcinogenic compounds or their metabolites and freeradicals generated by electromagnetic radiation. High sensitivitydetection of nucleoside adducts is of great importance in the evaluationof exposure of organisms to agents which modify nucleic acids. Examplesof nucleoside adducts include the reaction products of polycyclicaromatic hydrocarbons (PAH) at N2 of guanosine, aromatic amines andoxygen radicals at C8 of guanosine, of alkylating agents at N7 and O6 ofguanosine, and of mycotoxins at N7 of guanosine.

A preferred enzyme for use in the method is a polynucleotide kinaseenzyme (PNK) such as T4 polynucleotide kinase. This enzyme is widelyused in the preparation of ³² P 5'-dNMP, under standard reactionconditions of 37° C. at pH 8.5, by a reaction which involves thephosphorylation of 3'-dNMP with gamma³² P!-ATP and PNK. The inventorsattempt to phosphorylate 2',3'-dideoxyadenosine (which lacks3'-phosphate) with gamma³² P! ATP, using this enzyme and these reactionconditions, was not successful. Success can surprisingly however beachieved using lower temperature and/or lower pH conditions. Thepreferred temperature range is 4°-30° C. with 18° to 30° C. being morepreferred. The preferred pH range is 4.0-9.0. A high salt content, e.g.up to 150 mM NaCl, may be useful to promote the desired reaction.Incubation of the reactants under these conditions for times rangingfrom 10 minutes to 24 hours can give rise to good yields which increasewith longer reaction times. The use of phosphatase free T4 PNK in whichthe 3' phosphatase activity has been substantially removed may beadvantageous in eliminating side reactions which reduce the nucleosidemonophosphate yields.

Another enzyme that can be used in the method of the invention is aphosphotransferase enzyme extracted from barley seedlings (J. Biol.Chem., 257, No. 9, pp 4931-9, 1982). The enzyme has two activities: oneis the phosphotransferase which will transfer the phosphate from anucleoside-5'-monophosphate to the 5'-hydroxyl group of any othernucleoside, with a preference for purine deoxynucleoside phosphateacceptors. The enzyme has not, so far as is known, been previously usedin phosphorylating 2',3'-dideoxynucleosides. The other activity of thisenzyme is that the phosphoryl-enzyme intermediate can transfer thephosphate to water rather than the nucleoside acceptor, creatinginorganic phosphate. It is possible to control the enzyme activity byvarying pH, the ratio of donor to acceptor, and the addition of salts toremove the water available to the enzyme. Preferred conditions of useare 5° to 30° C. at pH 4 to 9, particularly 7 to 8.5. The methodrequires the introduction of 5'-NMP which is then used as the phosphatedonor. 5'-UMP is the preferred 5'-NMP.

Yet another phosphotransferase enzyme is derived from calf thymus.

A nucleotide analogue is a nucleoside analogue that has at least one5'-phosphate or 5'-thiophosphate group.

Nucleotide phosphate and thiophosphate donors are well known in thefield. Preferred examples of nucleotide phosphate donors are ATP,5'-UMP, ATP-gammaS, 5'-UMP-αS. These donors may be radiolabelled with ³²P, ³³ P and ³⁵ S so that the label transfers with a phosphate orthiophosphate group to the nucleoside analogue. A preferred nucleotidephosphate donor is gamma³² P! ATP. Reaction of this donor with a2',3'-dideoxynucleoside using PNK, gives rise to a 5'- α³² P!nucleosidemonophosphate.

These nucleoside monophosphates can be readily and efficiently convertedby known means to the corresponding triphosphate. By the use of ³³ P or³⁵ S, the corresponding α³³ P! or α³⁵ S!dideoxynucleoside triphosphatescan be made.

When using an enzyme to phosphorylate a nucleoside, it has been usualand useful to provide a large excess of the chosen nucleotide phosphate(or thiophosphate) donor, which has the effect of pushing the reactionin the desired direction. When the nucleotide phosphate (orthiophosphate) donor is radiolabelled, it is not practicable to providea large excess. As a result, the reaction conditions are more criticalif a good yield of a desired radiolabelled nucleotide is to be obtained.

In another aspect, this invention provides a kit for sequencing nucleicacids which kit comprises a supply of each of the four chain terminatingnucleotide or nucleotide analogue labelled with a radioisotope.Preferably the kit comprises α³² P! and/or α³³ P! and/or α35S! chainterminating nucleotide analogues e.g. dideoxynucleoside triphosphatestogether with a polymerase enzyme e.g. a T7 DNA polymerase, a supply ofeach of the four dNTPs and a buffer containing Mn²⁺. The provision ofthe labelled ddNTPs should make their use in sequencing reliable withimproved accuracy through reduced background and more even bandintensity.

In another aspect the invention provides a method of sequencing anucleic acid by a chain-termination technique, which method compriseseffecting template-directed enzymatic synthesis using as a chainterminator a nucleotide or nucleotide analogue labelled with aradioisotope and detecting products of enzymatic synthesis by means ofthe radioisotope.

In yet another aspect, the invention provides any one of ddCTP and ddGTPand ddTTP which is radiolabelled by ³² P or ³³ P or ³⁵ S wherein theradiolabel is preferably present in an α-phosphate group.

The detection of chain-termination DNA sequencing products afterseparation by gel electrophoresis has been achieved in any of severalways. The original methods involved the use of α-³² P! dATP tointernally-label the newly-synthesised DNA. Similarly, radiolabelledoligonucleotide sequencing primers can also be used. More recently,primers and nucleotides labelled with fluorescent dyes have also beenused with expensive, sensitive instruments which detect the fluorescentproducts. These methods work well only if care is taken to ensure thatall the DNA chains are correctly terminated by dideoxy-nucleotides. Anychains which terminate with deoxy-nucleotides at the 3' end maycontribute to background signal in the final electrophetogram. Suchterminations can occur when the polymerase is not highly processive orwhen the template contains strong secondary structures. Suchnon-specific stops are commonly seen in sequencing experiments andusually result in either errors or require re-sequencing to correctlyassign the affected bases.

There have also been several successful methods using fluorescent dyesattached to the chain-terminating dideoxynucleoside (Prober et al, Lee,L G, Connell, C R , Woo, S L, Cheng, R D, McArdle, B F, Fuller, C W,Halloran, N D & Wilson, R K, (1992), Nucleic Acids Res., 20, 2471-2483.)These methods have the advantage that the label is directly attached tothe molecule which causes chain-termination. The false or backgroundterminations, when they occur, will not be detected by thefluorescence-detection instrument. There are two drawbacks to thesemethods. One is that the dye-tagged dideoxynucleoside triphosphates arenot generally as efficient substrates for DNA polymerases as non-taggeddideoxynucleotides. They must be used at relatively high concentrations,and their rates of reaction vary with local sequence context giving riseto much less uniform band (or peak) intensities than non-taggednucleotides. The second is that the equipment used to detectfluorescent-tagged DNA is complex and expensive compared with theequipment needed for traditional autoradiographic detection.

This invention features the benefits of placing the detectable label onthe chain-terminating nucleotide without the drawbacks of expensivedetection equipment or reduced reactivity with DNA polymerase. This isdone by tagging the chain-terminating nucleotides with radioactiveisotopes, especially of sulphur or phosphorus. This requires theefficient production of all four labelled dideoxynucleosidetriphosphates and also requires a workable method to use them.

The original DNA polymerase used for chain-termination DNA sequencing(the large fragment of E. coli DNA polymerase I or Klenow enzyme) usesdideoxynucleotides relatively inefficiently. Most sequencing methodsusing this polymerase require the use of dideoxynucleotides atconcentrations up to 50 or 100 times higher than the concentration ofthe corresponding deoxynucleotide. Expressed as a concentration ratio,ddNTP:dNTP is as high as 100:1. The typical minimum amount of dNTP forpractical sequencing is on the order of 30 pmol each to allow extensionof 0.5 pmol of primed template by an average of 240 bases. Thus, 3000pmol of ddNTP may be required for sequencing with Klenow polymerase. Theminimum specific radioactivity for detection of extension products from0.5 pmol of template DNA with X-ray film and overnight exposure isapproximately 500 Ci/mol. While this is practical for ordinarysequencing methods with non-labelled ddNTPs, the high amounts requiredmake sequencing with labelled ddNTPs and this polymerase prohibitivelyexpensive, wasteful and hazardous, requiring as much as 1.5 mCi per laneof sequence.

A key feature of the new sequencing method is the use of a DNApolymerase which efficiently uses dideoxynucleoside triphosphates sothat the concentration ratio (and hence amount required) is reduced topractical levels. One such polymerase is modified T7 DNA polymerase whenused in the presence of Mn²⁺ (Tabor and Richardson, J. Biol. Chem. 264,6447-6458). With this polymerase, dideoxynucleoside triphosphates reactalmost as efficiently as deoxynucleoside triphosphates, allowing the useof a concentration ratio of ddNTP:dNTP of 1:100. This ratio is 10,000times more favourable for efficient use of dideoxynucleotides than theratio for Klenow polymerase. With this polymerase and the amounts oftemplate outlined above, as little as 0.3 pmol or 0.15 μCi of labelleddideoxynucleoside triphosphate will be required for each lane of thesequencing experiment. This amount is readily used economically andsafely. Other DNA polymerase enzymes which make efficient use ofdidoexynucleoside triphosphates can also be used for this sequencingmethod.

An additional benefit when using modified T7 DNA polymerase and Mn²⁺ isthe uniform band intensities obtained. This makes interpretation of thesequencing experiment more accurate.

The following examples illustrate the invention.

T4 Polynucleotide Kinase=5'-dephosphopolynucleotide5'-phosphotransferase EC 2.7.1.78

Polynucleotide kinase 3' phosphatase free=5'-dephosphopolynucleotide5'-phosphotransferase EC 2.7.1.78--from T4 am N81 pse T1 phage infectedE. coli BB

EXAMPLE 1

3.5 μmoles of each 2',3'-dideoxynucleoside (all four bases) wereindividually mixed with 50 units of 3'-phosphatase free PNK and 5 nmolesof gamma³² P! ATP in a buffer containing 50 mM Tris-HCl pH 7.5, 2.5 mMDTT and 30 mM Mg Acetate, 150 mM NaCl, 0.1 mM Spermine and 0.5 mM NH₄Cl. The final reaction volumes were 100 μl and the reactions wereincubated at 18° C. The reactions were followed by TLC analysis on PEIcellulose plates developed in 0.5M LiCl and 1M formic acid.

Table 1 shows the Conversions to ³² P!ddNMP's with time.

                  TABLE 1                                                         ______________________________________                                        TIME       %  .sup.32 P!                                                                         %  .sup.32 P!                                                                            %  .sup.32 P!                                                                       %  .sup.32 P!                             (minutes)  ddAMP   ddCMP      ddGMP ddTMP                                     ______________________________________                                        30         44.1    33.6       12.7  28.8                                      90         64.3    83.3       26.7  76.6                                      ______________________________________                                    

The TLC system was calibrated by using ³² PO₄, ³² P!ddAMP (producedchemically) and 5'dAMP as markers. This enabled the TLC plates to beinterpreted and the peaks, obtained using a beta-particle scanner,identified.

EXAMPLE 2

The 32P!ddNMP products from Example 1 were synthesised on a largerscale.

35 μmoles of each 2',3'-dideoxynucleoside (all four bases) wereindividually mixed with 500 units of 3'phosphatase free PNK and 75 mCi(25 nmoles) of gamma³² P! ATP in a buffer containing 50 mM Tris-HCl pH7.5, 2.5 mMDTT, 30 mM Mg Acetate, 150 mM NaCl, 0.1 mM Spermine and 0.5mM NH₄ Cl. The final reaction volumes were 2 ml and the reactions wereincubated at 18° C. for 2 hours. The reactions were followed by TLCanalysis on PEI cellulose plates developed in 0.5M LiCl and 1M formicacid.

After 2 hours the reactions were stopped by the addition of 2 mlabsolute ethanol. After filtration the reactions were purified by HPLCion-exchange chromatography. TLC analysis of the purified monophosphatesshowed that the ³² P!ddAMP contained some inorganic ³² PO₄. The otherthree monophosphates all had purities in excess of 90%. The yields ofthe reactions were of the same order as those seen in the small scaleassays in Table 1.

The ³² P!ddNMP's were converted to the respective α³² P!ddNTP's readilyand efficiently by standard methods.

After purification by HPLC ion-exchange chromatography the α³² p!ddNTP's were resuspended at ≈4 mCi/ml in aqueous solution. The finalyields from gamma³² P! ATP were:

ddATP 48%

ddCTP 46%

ddGTP 15%

ddTTP 24%

Samples were taken for identification by analytical HPLC against therespective non-radioactive ddNTP marker and for use in DNA sequencing.The results showed that with all four ddNTP's the radiolabelled α³²P!ddNTP and the non-radioactive ddNTP eluted from the HPLC column atexactly the same time. Also in the absence of any other terminator orradiolabel, apart from that synthesised above, the sequence of an M13template was successfully determined when compared to the sequenceproduced using α³⁵ S! dATP internal label and non-radioactive ddNTP's.

This proves that the α³² P! ddNTP's were made and therefore that PNKcan, under these conditions, phosphorylate 2',3'-dideoxynucleosides.

EXAMPLE 3 BARLEY SEEDLING PHOSPHOTRANSFERASE

0.5 μmoles of uridine-5'-monophosphate (5'-UMP) was mixed individuallywith 0.5 μmoles of each 2',3'-dideoxynucleoside (except 2',3'-ddG whichwas 0.15 μmoles of 5'-UMP and 2',3'-ddG due to solubility problems with2',3'-ddG--note still in 1:1 mole ratio) and 10 μl Barley SeedlingPhosphotransferase (1.3 units/ml) in 50 mM Tris-HCl pH 7.5, 1 mM MgCl₂and 0.002% Triton-100. The final reaction volume was 50 μl and thereaction was incubated at 25° C. for 4 hours. 15 μl samples were removedfor analysis by ion-exchange chromatography, the samples were made up to120 μl with water prior to loading. Analysis by this method showed thatall 4 base 2',3'-dideoxy nucleoside-5'-monophosphates had beensuccessfully made.

A second experiment, this time using 5'-UMP spiked with ³² P! 5'-UMP,under the same conditions as above except using a 5'-UMP:2',3'-dideoxyadenosine mole ratio of 24:1, gave a radiolabelled peak of2',3'-dideoxyadenosine-5'-monophosphate on an analytical HPLC systemwhich exactly matched that produced by the PNK method. The HPLC systemwas calibrated using a mixture of non-radioactive 5'-UMP and 5'-dAMP (noddAMP available commercially). These were shown to be well separated bythe system with the 5'-dAMP running slightly slower than the ³² P!2',3'-dideoxyadenosine-5'-monophosphate, which is to be expected on anion-exchange system due to the 3'-OH group. When this reaction wasrepeated at pH 5.0 in 50 mM sodium acetate buffer the HPLC analysisrevealed a very fast running radiolabelled peak that was identified asinorganic phosphate. This showed that the reaction is pH dependent. Theaddition of high concentrations of salt may also be beneficial andincrease the yield of monophosphate produced by inhibiting theproduction of inorganic phosphate.

EXAMPLE 4 SEQUENCING PROTOCOL

Sequencing was carried out using the Sequenase Version 2.0 kit from USBiochemical Co., Cleveland, Ohio.

1 μl (0.5 pmol) Primer, 2 μl reaction buffer, 5 μl (1 μg) controltemplate and 2 μl water were mixed in a clean sterile vial. This washeated to 65° C. for 2 minutes and then slowly cooled to 30° C. To thiswas added 1 μl DTT (0.1M) solution, 2 μl extension labelling mix(diluted 1:5), 1 μl Mn⁺⁺ buffer, 2 μl Sequenase DNA polymerase (diluted1:8) and 0.5 μl water (this was replaced by 0.5 μl α³⁵ S! dATP in theinternally labelled control, this also used the standard termination mixand not the one listed below). This was left at room temperature for 5minutes. 1 μl of a mix of all 4 dNTP's (either 30 μmolar or 480 μmolarsolution), 1 μl of the relevant α³² P! ddATP dilution (containing arange of specific activity dilutions with a varying chemical content of0.3, 4.8 or 48 pmoles. The ddNTP:dNTP ratio was varied between 1:10 and1:100) and 0.5 μl water was added to 3.5 μl of the above solution. Thiswas incubated at 37° C. for 5 minutes and then 4 μl of stop dye wasadded to each reaction tube. All the reaction tubes were heated at70°-80° C. for 5 minutes. 4 μl of each reaction was loaded onto astandard 6% polyacrylamide sequencing gel that had been pre-run for 40minutes. The gel was run at 45 mA until the first dye had run off thegel, ≈ 2 hours. The gel was then dried before exposure to film AmershamHyperfilm MP overnight. The results showed a much improved sequencingtrack with low background and even band intensities.

EXAMPLE 5 PHOSPHORYLATION OF 2'-DEOXYADENOSINE

0.3 mgs of 2'-deoxyadenosine were mixed with 30 units of T4 PNK and 8nmoles of gamma³² P! ATP in 50 mM Tris-HCl pH 7.5, 5 mM DTT and 14 mMMgCl₂. The final reaction volume was 100 μl and the reaction wasincubated at 24° C. The reaction was followed using the same TLC systemas in Example 1.

The results showed that the incorporation to ³² P! 5'dAMP was

Time 45 minutes=4%

Time 240 minutes=15%

Overnight=44%

EXAMPLE 6 (THIOPHOSPHORYLATION)

3.5 μmoles of 2',3'-dideoxyadenosine, adenosine and 2'deoxyadenosinewere individually mixed with 150 units 3' phosphatase free PNK and 85pmoles of gamma³⁵ S! ATP in a buffer containing 50 mM Tris-HCl pH 7.5,2.5 mM DTT, 20 mM Mg Acetate, 0.1 mM Spermine and 0.5 mM NH₄ Cl. Thefinal reaction volumes were 100 μl and the reactions were incubated at18° C. The reactions were followed by TLC analysis on PEI celluloseplates developed in 0.5M LiCl and 1M formic acid.

After a 21 hour incubation the reactions contained

8.3% Adenosine-5'-monothiophosphate ³⁵ S!

11.2% 2'-deoxyadenosine-5'-monothiophosphate ³⁵ S!

2.8% 2',3'-dideoxyadenosine-5'-monothiophosphate ³⁵ S!

EXAMPLE 7 (MODIFIED SUGARS)

3.5 μmoles of 3'-azidothymidine was mixed with 50 units 3' phosphatasefree PNK and 5 nmoles of gamma³² P! ATP in a buffer containing 50 mMTris-HCl pH 7.5, 2.5 mM DTT, 20 mM Mg Acetate, 0.1 mM Spermine and 0.5mM NH₄ Cl. The final reaction volume was 100 μl and the reaction wasincubated at 18° C. The reaction was followed by TLC analysis on PEIcellulose plates developed in 0.5M LiCl and 1M formic acid.

After a 3 hour incubation the reaction contained 22%3'-azidothymidine-5'-monophosphate ³² P!.

EXAMPLE 8 (BASE MODIFICATIONS)

3.5 μmoles of 7-deazaadenosine (tubercidin), 3-nitropyrole nucleosideand 5-nitroindole nucleoside (ref: D Loakes and D M Brown, NAR, 1994,Vol. 22, No. 20, pg 4039-4043) were individually mixed with 50 units 3'phosphatase free PNK and 5 nmoles of gamma³² P! ATP in a buffercontaining 50 mM Tris-HCl pH 7.5, 2.5 mM DTT, 20 mM Mg Acetate, 0.1 mMSpermine and 0.5 mM NH₄ Cl. The final reaction volumes were 100 μl andthe reactions were incubated at 18° C. The reactions were followed byTLC analysis on PEI cellulose plates developed in 0.5M LiCl and 1Mformic acid.

After a 3 hour incubation additional peaks were observed on the TLCscans. These were presumably due to the substrate as they were not seenin the absence of substrate.

63.0% 7-deazaadenosine-5'-monophosphate ³² P!*

16.1% 3-nitropyrolenucleoside-5'-monophosphate ³² P!*

23.9% 5-nitroindolenucleoside-5'-monophosphate ³² P!*

* These products were only seen in the presence of the substrate andwere not seen in the control experiments without substrate.

EXAMPLE 9

3.5 μmoles of each 2',3'-dideoxynucleoside (all four bases) wereindividually mixed with 1000 units 3'phosphatase free PNK and 60 to 100mCi of gamma³³ P! ATP (at ≈3000 Ci/mmole) in a buffer containing 50 mMTris-HCl pH 7.5, 2.5 mM DTT, 20 mM Mg acetate, 150 mM NaCl, 0.1 mMspermine and 0.5 mM NH₄ Cl. The final reaction volumes were 2 ml and thereactions were incubated at 18° C. from 2 to 6 hours. The reactions werefollowed by TLC analysis on PEI cellulose plates developed in 0.5M LiCland 1M formic acid.

The reactions were stopped by the addition of 2 ml absolute ethanol.After filtration the reactions were purified by HPLC ion-exchangechromatography. TLC analysis of the purified monophosphates showed thatthe ³³ P!ddAMP contained some inorganic ³³ PO₄. The other threemonophosphates all had purities in excess of 90%. The yields of thereactions were of the same order as those seen in the small scale assaysin Table 1.

The ³³ P! ddNMP's were converted to the respective α³³ P! ddNTP'sreadily and efficiently by standard methods.

After purification by HPLC ion exchange chromatography the α³³ P!ddNTP's were resuspended at ≈4 mCi/ml in aqueous solution. The finalyields from gamma³³ P! ATP were:

ddATP 40%

ddCTP 30%

ddGTP 20%

ddTTP 16%

Samples were taken for identification by analytical HPLC against therespective non-radioactive ddNTP marker and for use in DNA sequencing.The results showed that with all ddNTP's the radiolabelled α³³ P! ddNTPand the non-radioactive ddNTP eluted from the HPLC column at exactly thesame time.

EXAMPLE 10 SEQUENCING DNA

Using the methods outlined in Examples 1-3, α³² P! ddGTP, α-³² P! ddATP,α-³² P! ddTTP and α-³² P! ddCTP were prepared with a specific activityof approximately 2000 Ci/mmol and concentration of 0.5 μM. These wereused in the following fashion to determine the base sequence of M13mp18DNA. Many of the reagents described here can be found in the SequenaseDNA sequencing kits produced by US Biochemical Co., Cleveland, Ohio.

Four nucleotide termination mixes were prepared by mixing 2 μl of 15 μMdATP, dTTP, dCTP, dGTP and 100 mM NaCl with 0.6 μl (containing 0.3 pmol)of each of the radiolabelled ddNTP solutions.

Template DNA (M13mp18, 1.0 μg in 5 μl) was mixed with 0.5 pmol (1 μl) ofM13 "-40"23-mer oligonucleotide primer, 1 μl of MOPS buffer (400 mMmorpholinopropanesulphonic acid-NaOH, pH 7.5, 500 mM NaCl, 100 mM MgCl₂,1 μl of Mn buffer, 50 mM MnCl₂, 150 mM Isocitrate, sodium salt) and 2 μlof water for a total volume of 10 μl. This mixture was warmed to 37° C.for 10 min to anneal the primer to the template. The mixture was chilledon ice and 1 μl of 0.1M dithiothreitol and 2 μl of polymerase mixture(1.6 Units/μl Sequenase Version 2.0 T7 DNA polymerase (U.S. BiochemicalCorp.), 2.0 Units/ml inorganic pyrophosphotase 20 mM Tris.HCl pH 7.5, 2mM DTT 0.1 mM EDTA, 50% Glycerol) added and mixed well. Then 3 μlportion of this DNA and polymerase mixture were mixed with thepre-warmed (to 37° C.) termination mixtures 2.6 μl) described above. Themixtures were allowed to incubate for 10 min at 37° C., then 4 μl ofstop solution (95% Formamide 20 mM EDTA 0.05% Bromophenol Blue 0.05%Xylene Cyanol FF) were added to stop the reaction.

The mixtures were heated briefly and applied to a denaturingpolyacrylamide electrophoresis gel buffered with Tris-taurine-EDTAbuffer (U.S. Pat. No. 5,134,595, Pisa-Williamson, D. & Fuller, C. W.(1992) Comments 19, 29-36). After electrophoresis, the gel was dried bystandard procedures and exposed to film overnight. The resulting DNAsequencing autoradiogram was exceptionally free of background, clearlyshowed the identity of the first nucleotide added to the 3' end of theprimer, had uniform band intensities.

EXAMPLE 11 SEQUENCING USING dITP TO ELIMINATE COMPRESSION ARTIFACTS

Compression artifacts occur when the DNA being separated on a sequencingelectrophoresis gel are not completely denatured. Nucleotide analoguessuch as dITP (deoxyinosine triphosphate) which replace dGTP in thesequencing reactions can eliminate compression artifacts (Tabor. S. andRichardson, C. C. (1987) Proc. Nat. Acad. Sci. USA 84, 4767-4771).Sequencing reactions were run exactly as described in Example 10 exceptthat the four nucleotide termination mixes were prepared by mixing 2 μlof 75 μM dITP, 15 μM dATP, dTTP, dCTP and 100 mM NaCl with 0.6 μl(containing 0.3 pmol) of each of the radiolabelled ddNTP solutions.Sequencing of M13mp18 template DNA was done using a different primerchosen to sequence through a region prone to compression artifacts. Whenthe dITP-containing mixture was used, no compression artifacts wereobserved while control sequences run with dGTP mixtures did havecompressed, unreadable regions.

We claim:
 1. A method of making a nucleotide, nucleotide analogue, ornucleotide adduct, having a 5═-phosphate or a 5'-thiophosphate groupsaid method comprising reacting a starting nucleoside, nucleosideanalogue, or nucleoside adduct having a 5'-OH group but no 3'-phosphategroup, with a nucleotide phosphate donor or nucleotide thiophosphatedonor in the presence of an enzyme which catalyses the reaction.
 2. Amethod as claimed in claim 1 wherein the nucleotide phosphate donor ornucleotide thiophosphate donor is radiolabelled with a radioisotopeselected from the group consisting of ³² P, ³³ P, and ³⁵ S, whereby theobtained nucleotide, nucleotide analogue, or nucleotide adduct isradiolabelled at the 5'-phosphate or 5'-thiophosphate group with ³² P,³³ P, or ³⁵ S.
 3. A method as claimed in claim 1 wherein the startingnucleoside analogue is a 2',3'-dideoxynucleoside.
 4. A method as claimedin claim 1 wherein the starting nucleoside analogue is selected from thegroup consisting of 3'-fluoro, 3'-amino, and 3'azido nucleosides,polyamide nucleic acids and an anti-sense oligonucleotide.
 5. A methodas claimed in claim 1 wherein the enzyme is a polynucleotide kinase. 6.A method as claimed in claim 1 wherein the reaction is performed at4°-30° C. and pH 4-9.
 7. A method as claimed in claim 1 wherein thenucleotide phosphate donor or nucleotide thiophosphate donor is selectedfrom the group consisting of gamma³² P-ATP, gamma³⁵ S-ATP, and gamma³³P-ATP.
 8. A method as claimed in claim 1 wherein a2'-3'-dideoxynucleoside is reacted with gamma³² P-ATP to make a 5'-³² Pnucleoside monophosphate.
 9. A method as claimed in claim 8, wherein the5'-³² P nucleoside monophosphate is subsequently converted to the di- ortri-phosphate.
 10. A method as claimed in claim 1 wherein the enzyme isa phosphotransferase enzyme derived from barley seedlings.
 11. A kit forsequencing nucleic acids, which comprises each of the four chainterminating nucleotides or nucleotide analogues, wherein the chainterminating nucleotides or nucleotide analogues are labelled with aradioisotope.
 12. A kit as claimed in claim 11, wherein the chainterminating nucleotides or nucleotide analogues are dideoxynucleotides.13. A kit as claimed in claim 11 wherein each of the four chainterminating nucleotides or nucleotide analogues are labelled with aradioisotope selected from the group consisting of ³² P, ³³ P, and ³⁵ S.14. A kit as claimed in claim 11 wherein the kit further comprises apolymerase enzyme, deoxyadenosine triphosphate, deoxyguanosinetriphosphate, deoxycytidine triphosphate, and deoxythymidinetriphosphate.
 15. A kit as claimed in claim 14, wherein the polymeraseenzyme is a T7 DNA polymerase and the kit further comprises a buffercontaining Mn²⁺.
 16. A method of sequencing a nucleic acid by achain-termination technique, which method comprises effectingtemplate-directed enzymatic synthesis using as a chain terminator, anucleotide or nucleotide analogue labelled with a radioisotope anddetecting products of enzymatic synthesis by means of the radioisotope.17. A method as claimed in claim 16, wherein the enzymatic synthesisreaction is performed using a T7 DNA polymerase enzyme in the presenceof all four dNTPs and of the labelled chain terminating nucleotide ornucleotide analogue in a buffer containing Mn²⁺.
 18. A method as claimedin claim 16 wherein the radioisotope is ³² P or ³³ P or ³⁵ S.
 19. Achain terminator selected from the group consisting of ddCTP, ddGTP andddTTP which is radiolabelled with a member selected from the groupconsisting of ³² P, ³³ P and ³⁵ S.
 20. The chain terminator according toclaim 19 wherein the radiolabel is present in an α-phosphate group.